provided in skeletal muscle and the heart as revealed by the relatively large

Transcription

1 Journal of IPhysiology (1991) pp WT'ith 12 figures Printed int Great Br itaini ELECTRICAL RESPONSES OF CORONARY ARTERY SMOOTH MUSCLE ASSOCIATED WITH THE CARDIAC MUSCLE ACTION POTENTIAL IN THE MONKEY BY F. MEKATA From the Iepartment of Physiology, Primtate Research Institute, Kyoto University, Inuyama 484, Japan (Received 28 August 1990) SUMMARY 1. In cardiac strips of the monkey ventricle which included a section of coronary artery (cardiac preparation), a depolarizing response, which appeared to be an excitatory junction potential (EJP), and a hyperpolarizing response were observed from the coronary artery smooth muscles when an action potential was generated in cardiac muscle by the application of electrical stimulation to the cardiac muscle alone. 2. In an isolated preparation of the coronary artery dissected from the ventricle (arterial preparation), similar responses, an EJP and a hyperpolarizing response, could be evoked by electrical stimulation. 3. In the cardiac preparation, the threshold of electrical stimulation for generation of the cardiac action potential and for production of an electrical response in the vascular smooth muscle were the same. 4. The EJP and the hy7perpolarizing responses of the smooth muscle of the coronary artery ceased or were weakened by elimination of the adventitial connective tissues and endothelium. respectively, in both the cardiac preparation and the coronary artery preparation. 5. These results indicate that the action potential of cardiac muscle generated by electrical stimulation activates the nerves and the vascular endothelium, which, in turn, produce an EJP and a hyperpolarizing response of the coronary artery smooth muscle, respectively. INTRODUCTION Tension of the smooth muscle of blood vessels is under the control of both the nerves and the endothelium. Because a number of blood vessels are running within organs, the vascular smooth muscles may also be influenced by the tissues of each organ (Keatinge & Harman. 1980). Three possible routes, electrical, mechanical and chemical, could transmit signals from the tissue to the blood vessels. In most organs which are composed of non-excitable cells, the influence from the tissue is likely to be due to the release of chemical substances. Large local circuit current would be necessary to obtain significant electrical transmission. Such conditions can be provided in skeletal muscle and the heart as revealed by the relatively large.is 8755

2 240 F. MEKATA amplitude of extracellular potentials that can be recorded. Another organ composed of excitable cells is the brain. However, in the brain, the networks of neurons are so complex that local circuit current induced by nerve action potentials spreads generally at random and without direction; therefore it is doubtful that local currents work to adjust vascular tone. In reproductive organs and digestive ducts, the potential recorded extracellularly is very small (Bozler, 1938), perhaps because the ratio of intracellular and extracellular resistances in the longitudinal direction is small, suggesting that currents evoked by these smooth muscles are less likely to have an effect than activity in heart and skeletal muscle. It is known that changes in heart rate influence coronary blood flow by means of mechanical factors, such as the squeezing effect of the contracting myocardium, and also by chemical factors such as metabolites produced by the myocardium during contraction (Berne & Levy, 1981), but these factors do not account for all the behavioural characteristics of coronary blood flow under physiologically normal or abnormal conditions. Another possible route by which the myocardium could influence the coronary artery is through currents. Action potentials of cardiac muscle are synchronized so well that the muscle may behave as a battery large enough to have an effect on coronary arteries. Measurement of parameters such as coronary flow, diameter of the artery or its tension provide no direct evidence as to whether activation of cardiac muscle influences coronary artery smooth muscle, since these measurements are influenced by change in heart volume and aortic pressure. The aim of the present study was to clarify whether activation of ventricular muscle has any effects on coronary artery smooth muscle. The study was carried out on muscle strips from the ventricles which included the coronary artery, and from which muscle electrical behaviour was recorded. METHODS Sixty Japanese monkeys of either sex, 3-12 years old and weighing 3-14 kg, were used. Monkeys were killed according to following protocol. (1) Ketalar (10-15 mg (kg body weight)-') was administrated into the femoral or brachial muscle of the animal in a cage. (2) After the animal was anaesthetized fully, it was transferred onto the dissecting table, and Nembutal (15-20 mg (kg body weight)-') was administered intravenously. (3) Bilateral cervical arteries were exposed and dissected for exsanguination. (4) After at least 3 min following the initiation of exsanguination, dissection was started for organ sampling. Studies were performed on coronary arteries, 1-2 mm in outside diameter, in two preparations, a cardiac preparation and an arterial preparation. Cardiac preparation. Cardiac muscle strips, about 3-4 cm in length and about 1 cm in width, were removed from a part of the left ventricle, in which a large branch of the circumflex coronary artery runs within the ventricle tissue. The muscle strips were kept in standard Krebs solution at 36 C for about 1 h before the start of the experiments. A part of the cardiac preparation, about 1 cm from the end of the strip and about 2 mm in thickness, was dissected from the surface to remove the coronary artery from this region. The preparation was mounted in an organ bath. The cardiac muscle without the artery was pulled through holes in the stimulating plates which divided the organ bath into three compartments as shown in Fig. 1. The preparation was mounted so that the part containing the artery was in the left compartment of the organ bath with the left stimulating partition near the centre of the dissected part. A cut was made in the tissue of the cardiac muscle in the left compartment, about 0 5 mm in length along the long axis of the coronary artery, to expose a part of the artery within the ventricular muscle, to allow a microelectrode to be inserted into the smooth muscle of the coronary artery. To produce an action potential in the cardiac muscle, brief pulses of electrical stimulation were applied to the preparation through two needle electrodes placed 3 mm from the insulating plate in the left compartment and another electrode

3 INTERACTION BETWEEN HEART AND CORONARY MUSCLE 241 placed in the centre compartment ( JL 2 in Fig. 1). In the ventricles, nerves run along the coronary vessels and large and middle coronary arteries are undoubtedly innervated. However, there are conflicting reports as to whether fine coronary arteries and the arterioles are innervated (Feigl, 1983) or not (Henry & Meehan, 1971). In order to avoid the brief pulse directly stimulating the Microelectrode 1 Needle electrode Stimulating partition Coronary artery Ventricle Left compartment Centre compartment l Right compartment Fig. 1. Stimulating and recording arrangements for the preparation in the organ bath. Prolonged current (itl1) was applied through two silver plates. Membrane potentials of the smooth muscle and the cardiac muscle cells were recorded from the left compartment. For the brief pulse stimulation (._IFL 2)' two silver needle electrodes were used. The cardiac preparation was cut as shown in the figure and mounted through a small hole on the silver plate which divided the left and centre compartments. The artery is shown by the dashed line. The plate described as stimulating partition in the text is the left one of the two. Direction of flow of the solution is shown by short arrows. nerves, cardiac preparations were used in which there were no large- or middle-sized coronary arteries running in the longitudinal direction within the dissected part. The absence of such coronary arteries in this part was confirmed by viewing cross-sections under a microscope at a magnification of 40 x. Furthermore, data from cardiac preparations in which the electrical response of the arterial smooth muscle to three brief pulses applied within ms was recordably larger than that to a single pulse were not used, since this could indicate direct activation of nerves. Arterial preparation. This was prepared from a careful dissection (to protect the nerves) of a large branch of the left circumflex coronary artery which was similar in size to that of the cardiac preparation. The artery was mounted in the three compartments of the organ bath and the membrane potential was measured from the left compartment. In order to stimulate the nerves and endothelium of the arterial preparation, electrical stimulation was applied to the tissue through the needle-electrode arrangement described above. The size of the holes in the stimulating partition dividing the left and the centre compartments was adjusted to make the stimulation effective. Intracellular potentials of cardiac and smooth muscles were recorded from the left compartment. To assess the passive properties of the smooth muscle cell, a rectangular current, 1-5 s in duration, was applied through the two stimulating partitions (ii 1 in Fig. 1) to the arterial preparation. In order to confirm that the extracellular potential was negligible, after the intracellular recording had been made from the smooth muscle of the cardiac preparation the electrode was withdrawn and used to record the surface potential under the same conditions. Preliminary experiments showed that EJPs were eliminated by tetrodotoxin (TTX; 10-7M) with supramaximal effect. In studies described in Results, concentrations (3 x 10-6M) of TTX more than that usually needed to block nerve conduction were used in order to observe simultaneously that TTX does not block the hyperpolarizing response in addition to suppressing the EJP.

4 242 F. MECKATA Irrigation by the solutions in the organ bath and the arrangements for stimulating and recording electrical responses were the same as those described previously (Mekata, 1984) and explained in Fig. 1. Standard Krebs solution of the following composition was used (mm): Na', 134 4; K+, 5-9; Mg2+, 1-2; Ca2+, 2-5; Cl-, 1340; HCO3-, 15 5; H2PO4, 1 2; glucose, RESULTS The passive electrical properties of the -smooth muscle of the arterial preparation The resting membrane potential was stable and no spontaneous change in the potential was observed, as described previously for spiral strips of the same artery (Mekata, 1986). An action potential could not be induced even by an extremely strong outward current. The mean value of the membrane potential, mv (mean+ S.D.; n = 107), was slightly smaller than that recorded from spiral strips (Mekata, 1986). The values of the space and time constants were obtained by conventional analysis of electrotonic potentials evoked by the applied currents, using the spatial decay of the amplitude of the electrotonic potential and the spatial change of the time required to reach half-amplitude, respectively. The mean value of the space constant of mm (n = 5) obtained from this preparation was smaller than that measured previously in spiral strips (Mekata, 1986). This difference may be due to a difference in the angle of the long axis of the muscle cells with that of the muscle preparation. The mean value of the time constant was ms (n = 5). The steady-state voltage-current (V-I) curve demonstrated strong outwardgoing rectification, whereas the relationship was nearly linear in the hyperpolarizing direction. As the membrane was depolarized below -40 mv, the membrane resistance decreased markedly. The V-I curve was similar to that observed from other quiescent types of vascular smooth muscle (carotid artery and aorta, Mekata, 1971; Mekata, 1974). Responses of the arterial preparation to brief electrical pulses When a single brief pulse was applied to the artery through two Ag-AgCl needle electrodes, the smooth muscles within 3 mm of the stimulating partition showed one of three types of response: a depolarizing response (six of twenty-four preparations) with amplitude between 5 and 15 mv, a hyperpolarizing response (ten of twentyfour preparations) with amplitude between 5 and 15 mv, or a mixed response having both the depolarizing and the hyperpolarizing elements (eight of twenty-four preparations) (Figs 2 and 3). In the region more than 4 mm from the stimulating partition only the depolarizing response was recorded. The hyperpolarizing response decayed with distance from the partition and, in most cases, was not recordable beyond 4 mm from the partition. Production of the depolarizing response could be seen occasionally even 30 mm from the partition. Spatial decay of the amplitude of the depolarizing response was not clearly detected within a distance between 5 and 30 mm from the stimulating partition, which varied with each preparation. Beyond this distance, however, recording of a depolarizing response became impossible in most cases. This failure may be due to nerve damage. In these results and the results shown in later sections, it is clear that the depolarizing responses are EJPs. The times required for the maximum EJP and hyperpolarizing response to develop after a single brief pulse was applied were ms (n = 70) and 510 ±

5 INTERACTION BETWEEN HEART AND CORONARY MUSCLE ms (n = 95), respectively. When pulse stimulations were increased in duration from 10 to 100 /ts by increments of 10 gcs, or in intensity from 10 to 100 V by increments of 10 V, a threshold was reached after which the amplitude of both responses increased abruptly. However, neither was all or nothing, but rather a ps duration 80 V intensity -. uj X V intensity 140,s duration u \.A. -_A..\_m ps duration 100 V intensity % M x_ V intensity 100 ps duration %. M\00_00 Fig. 2. Depolarizing responses (EJPs) and hyperpolarizing responses of two separate arterial preparations to single brief pulse stimulations. The first and third lines show the effect of increasing the pulse duration at constant intensity, and the second and fourth lines show the effect of increasing the stimulus intensity at constant pulse width. The first and second lines were obtained from one arterial preparation and the third and fourth lines were obtained from another. The former was recorded at 5 mm from the stimulating partition and the latter at 05 mm from the partition. gradual increase was seen, suggesting non-uniformity within nerve fibres (Fig. 2). The thresholds for the duration and the intensity showed large interpreparational variations. Application of repetitive brief pulses at ms intervals caused graded increases in the amplitudes and durations of both responses as the number of brief pulses increased, demonstrating summation of responses. When repetitive pulses were applied at intervals longer than about 1 s for both responses, summation ceased. Facilitation was not seen for either the EJP or the hyperpolarizing response. Both EJP and hyperpolarizing responses showed a reduction in amplitude for pulses with long intervals as shown in Fig. 3. The effect of pulse duration was investigated, to determine the times required to reach a maximum deflection for the EJP and the hyperpolarizing responses, by applying single pulses having 01 and 10 ms durations. The responses produced by two pulse width were directly compared in each preparation. For the EJP, the time 1 s

6 244 F. MEKATA to reach maximum deflection increased by % (n = 6) ( ms for 01 ms pulse and ms for the 10 ms pulse). For hyperpolarizing responses, the time to reach maximum deflection decreased by % (n = 5) ( ms for the 01 ms pulse, and ms for the 10 ms pulse). Single shock shocmkr Three shockspu ms 1 s 5 s,- - -_ Single shock 10 Three shockss _ 300 ms _%A-o_ ~ -~ "Wl-~ 1 s W _~~~ 110 mv Fig. 3. EJPs (upper records) and hyperpolarizing responses (lower records) of the arterial preparations to a single shock and three repetitive shocks of the same intensity. In the upper panel, the stimulus intensity was 30 V and the pulse duration 70 /is and in the lower panel 50 V and 100,us. The interval between pulses is shown over each trace. Upper and lower traces were obtained at 3 and 0 5 mm respectively from the stimulating partition, and from different preparations. When the arterial preparation was in the left compartment of the organ bath and was not passed through the hole in the partition, brief pulses failed to evoke any electrical responses from the coronary arteries. Electrical responses of ventricular muscle and smooth muscle of the coronary artery in the cardiac preparation to brief electrical pulses Each experiment on the cardiac preparation started with a recording of the action potential of the ventricular muscle generated by a single pulse. The pulse duration was increased from 10 to 140,us by increments of 10,us and increased in intensity

7 INTERACTION BETWEEN HEART AND CORONARY MUSCLE 245 from 10 to 100 V by increments of 10 V, while fixing either the voltage or the duration. As the stimulation reached its thresholds for intensity and duration, the ventricular muscle generated action potentials in an all-or-nothing manner (Fig. 4). The ranges of the thresholds were ,ts for the duration and V for the intensity, and showed large interpreparational variation. A ,s duration 30 V intensity - _ N _ V intensity _E loo1100 mv.a 40,us duration _ horny B 1 s 50 V intensity ,us duration 50,us duration V intensity \~ ~ jo 100mV 1 s Fig. 4. In a cardiac preparation, responses of the ventricular muscle (upper trace) and coronary artery smooth muscle (lower trace) to a single brief pulse. One example in which depolarizing response (EJP) (A) and hyperpolarizing response (B) were observed in the smooth muscle is shown here. Upper records, the effect of increasing the pulse duration at constant stimulus strength. Lower records, the effect of increasing stimulus intensity at constant pulse duration. The electrical responses of the coronary artery smooth muscle of the cardiac preparation to brief pulses were then recorded. The smooth muscle cells were found to be electrically quiescent. The mean value of membrane potential was

8 246 F. MEKATA mv (n = 128), which did not differ from that recorded from the arterial preparations. Cardiac muscle generated its action potential upon the application of a single brief pulse. Two kinds of electrical response were recorded from the cell membrane of the arterial smooth muscle (Figs 4 and 5). One of the responses was a phasic depolarizing deflection of the membrane potential, an EJP, and the other was a hyperpolarization. The responses were not always both generated in the same cell. The depolarizing responses alone (nineteen or thirty-two preparations) was more frequently observed than the hyperpolarizing response alone (one of thirty-two) or the mixed response (twelve of thirty-two). The EJP took ms (n = 79) to peak and had an amplitude between a few and 20 mv. The hyperpolarizing response took ms (n = 52) to peak and had an amplitude between a few and 15 mv. The time courses of both responses were similar to those seen when the arterial preparation was stimulated by three brief pulses or a single prolonged pulse rather than by a single brief pulse. When the duration or the voltage reached threshold, the EJP and the hyperpolarizing response had a maximum amplitude. Increasing the duration or the voltage above threshold did not change the amplitude of the response. In general, these responses were evoked in a clearly all-or-nothing manner. Neither response was observed below the threshold of electrical stimulation for the generation of a cardiac-muscle action potential. In the cardiac preparation, the thresholds for the duration and intensity for the EJP and the hyper-polarizing response of the smooth muscle were the same as those for generation of the action potential in the ventricular muscle, in spite of large interpreparational variation of the threshold. The electrical responses of both the cardiac and smooth muscle were recorded when three brief pulses, which were slightly stronger than the threshold, were applied at various intervals between 10 ms and10 s (Fig. 5). Records from the ventricular muscle showed an absolute refractory period of about 300ms. Only a single action potential was evoked when three shocks were applied with intervals shorter than 100ms. The smooth muscle also responded to three shocks with shorter intervals than 100ms, with simple phasic electrical change having one peak for the EJP or the hyperpolarization, the amplitude of which equalled that produced by a single shock. This behaviour of the arteries in the cardiac preparation differed from that of the artery in the arterial preparation. If three pulses were applied with an interval of 1 s, the cardiac muscle generated three action potentials in a completely normal manner and the arterial smooth muscle of the cardiac preparation showed electrical responses with three peaks. If repetitive brief pulses having comparatively long intervals of 3-10 s were applied, the amplitude of the hyperpolarizing response decreased gradually (Fig. 5), though the EJP showed little change in most cases or occasionally an increase in amplitude. Such changes were not seen in the records of the action potential of the ventricle muscle. These facts provide strong evidence that the EJP and the hyperpolarizing response were real transmembrane potentials of the smooth muscle cell membrane, and not extracellular potentials. When the cardiac preparation was cut through completely within1 cm of the stimulating partition which divided the left and centre compartments of the organ bath, an electrical response was not evoked either in the cardiac muscle or in the smooth muscle, even by stimuli much stronger than the threshold measured before cutting (Fig. 6).

9 INTERACTION BETWEEN HEART ANYD CORONARY MUSCLE 247 Once the EJPs to brief pulses had been recorded, the artery was dissected from the cardiac tissue and replaced in the same position, which resulted in weakening of the connection between the cardiac muscle and the coronary artery. No electrical responses to brief stimulation were recorded from the dissected artery, although the A Single shock Three shocks 300 ms 5 s interval 10mV o 1s B Single shock ms Three shocks 500 ms 5 s interval Xt X X X 1100 mv %\I00 \_s_ 120 mv 1 s Fig. 5. Effect of repetitive brief pulse stimulation on the ventricular muscle (upper traces) and coronary artery smooth muscle (lower traces), in the cardiac preparation. The interval between shocks is shown above the traces. Examples are shown in which the smooth muscle responded with an EJP (A) or a hyperpolarizing response (B) to pulse stimulation.

10 248 F. MEKATA \J\k- 100 mv (ventricle) Control L 10 mv (coronary) After cutting - is b Fig. 6. Effect of cutting the tissue on the electrical response of the cardiac preparation to brief stimulation. The action potential of the ventricle muscle and the EJP of the arterial smooth muscle associated with brief pulse stimulation were recorded from points a and b on the ventricle muscle (left and centre) and from point c on the smooth muscle (right). The ventricular muscle of the cardiac preparation was cut transversely about 1 cm from the stimulating partition, as indicated by the arrow. Upper traces, records from intact preparation; lower traces, records after cutting. J \ ~~~~~~~~~~10 mv Fig. 7. Effect of separation of the coronary artery from the ventricular tissue on.the depolarizing response. The EJPs of the smooth muscle of the coronary artery evoked by a single brief pulse having the same intensity and duration were recorded from an intact cardiac preparation (left), after separating the coronary artery (centre) and after the artery was returned to the ventricular tissue (right), and pressed down softly onto it. cardiac muscle generated an action potential in response to stimulation. When this artery was softly pressed down to make close contact between the two different tissues, some EJPs were again observed (Fig. 7). These results suggest that brief pulses do not stimulate coronary artery smooth muscle or nerves directly but first induce action potentials in cardiac muscle which in turn evoke an electrical response in coronary artery smooth muscle in a direct or indirect way. In all excitable tissues, the generation of action potentials produces local circuit currents which depend mainly on the electrical resistances of the extracellular and intracellular solutions. A part of the EJP or the hyperpolarization recorded 1 s

11 INTERACTION BETWEEN HEART AND CORONARY MUSCLE 249 intracellularly from the arterial smooth muscle of the cardiac preparation on generation of an action potential in cardiac muscle may reflect the deflection of extracellular potentials. Thus, extracellular potentials were measured at three to five different distances (0, 10, 20, 40 and 80,tm) from the cell surface of the arterial A 150 mv B C D 'I E 20 mv Fig. 8. Records of extracellular and intracellular potentials from the arterial smooth muscle cells when a single brief pulse was applied to the cardiac preparation after the solution level in the organ bath was lowered. A, intracellular potential of the ventricle muscle cells. B, intracellular potential of the smooth muscle cells. C, extracellular potential recorded on surface of the smooth muscle cell. D, real transmembrane potential obtained from the difference between B and C. smooth muscle after the microelectrode was withdrawn from the cells once the intracellular potential had been recorded. Under standard conditions of the organ bath, only small potential deflection amplitudes were recorded at the initiation of the cardiac action potential. The potential deflection was larger nearer to the cell surface but had a maximum of only 2 mv even at the surface, which in most preparations was less than 10 % of the intracellular potential deflection. No recordable potenfial deflection was observed 100 /am from the surface. These results indicate that in the recording system of the present experiments, the potential recorded intracellularly from the smooth muscle cell was approximately equal to the transmembrane potential. However, when the solution level in the organ bath was lowered so that there was a large increase in the electrical resistance of the extracellular space, the external potential due to local circuit current evoked by the cardiac action potential attained a maximum larger than 30 mv (Fig. 8). Effects of tetrodotoxin and the elimination of adventitial tissue and endothelium The effects of TTX on electrical responses of the smooth muscles evoked by brief pulses were investigated in arterial preparations producing an EJP (n = 5) or a mixed response (n = 4). Intracellular recordings were made at 1-5 and 0 5-0'7 mm from the stimulating partition respectively. Single brief pulses or three pulses with is

12 250 }X JIE.4 TA short breaks (10-20 ms) at 3 min intervals were applied. One example in which a distinct effect was obtained is shown in Fig. 9. When TTX (3 x 10-6M) was applied, the EJP ceased almost completely within 10 min. The response recovered partially following removal of TTX. The amplitude of the hyperpolarizing response, however, Control TTX (5 min) TTX (8 min) Recovery I10mV Fig. 9. Effect of TTX, (3 x 10-6M) on the EJP and the hvperpolarizing response evoked by a single brief pulse in the arterial preparation. Records show changes in the responises 5 and 8 min after application of TTX and 10 miii after washing out. Records at 0 7 mm from the stimulating partition. increased after the application of TTX by % (n = 4). The suppressing effect of TTX on the EJP was observed in regions both near ( mm) and far ( mm) from the stimulating partition. These results suggest that the EJP recorded during the present experiments is initiated by nerve stimulation resulting from the electrical shock, but not by direct stimulation of the nerve terminals. In the arterial and cardiac preparations, adventitial connective tissues on the upper surface of the arteries were carefully removed using forceps, from a region of about 05 cm surrounding the recording point of the microelectrode. After this treatment, the membrane potentials measured from the smooth muscle of the arteries in both preparations had similar values to those before the treatment. The treatment strongly suppressed the EJP of the arterial smooth muscle both in the cardiac preparation (control, 13+4 mv; without adventitia, 3+2 mv; n = 7) and the arterial preparation (control, 10+2 mv; without adventitia, 2+2 mv; n = 8) (Figs 10 and 12). However, for the hyperpolarizing response, little or no decrease in the amplitude was seen after treatment in either preparation. Elimination of the endothelium, which was accomplished by inserting a wire, led to a reduction in the hyperpolarizing response induced by brief pulses in both the cardiac (control, 12+3 mv; without endothelium, 4+2 mv; n = 5) and arterial (control, mv; without endothelium 2 +1 mv; n = 8) preparations (Figs 10 and 12). The EJP did not change in the arterial and the cardiac preparations after this treatment. In order to obtain evidence that the insertion of the wire was useful for the removal of the endothelium, the effect of the application of acetylcholine, which can induce endothelium-derived hyperpolarization of some vascular smooth muscle (rabbit aorta, Beny & Brunet, 1988; canine mesenteric artery, Komori, Lorenz & Vanhoutte, 1988; rabbit saphenous artery, Komori & Suzuki, 1987) on the hyperpolarizing response evoked by a single brief pulse ( ms duration) was investigated in the arterial preparation. Acetylcholine (10 jug ml-') induced significant hyperpolarization (21 +4 mv; n = 8) of the smooth muscle cell membranes in all examples of 2 s

13 INTERACTION BETWEEN, HEART AiND CORONARY MUSCLE 251 intact preparations (Fig. 10). In the arterial preparations which were treated by the wire, acetylcholine (10 tg ml-') caused only a small hyperpolarization (2+2 mv; n = 5). These results suggest that the EJP and the hyperpolarizing response of the smooth muscles of the arteries of the cardiac preparations were evoked by A Control _% Without connective tissue J20 mv 1 s B Control Without endothelium 120 mv C Control U - - -~~~ Without endothelium 120 mv Fig. 10. Arterial preparation. A, effect of the removal of the connective tissue on two different types of electrical responses, the EJP (left) and mixed (right) responses, evoked by a single brief pulse. Left and right were recorded 3 and 0-5 mm from the stimulating partition, respectively. B, effect of elimination of endothelium on the hyperpolarizing (left) and mixed (right) responses. These were both recorded 0 5 mm from the partition. (1, upper record shows electrical change produced about 1 min after application of acetylcholine (10 jug ml-') in the arterial preparation with endothelium. Lower trace, effect of acetylcholine (1O,ug ml-') on the preparation without endothelium. Records at 2-0 mm from the stimulating partition. activations of the nerve and endothelial cells, respectively, and were stimulated by the cardiac action potential. Adventitial connective tissue of the arterial preparations were removed from a region of about 0 5 cm surrounding the stimulating partition. After this treatment, a strong single pulse or three repetitive brief pulses failed to evoke the EJP either in

14 252 F. MEKATA a b I m a b c di IY -F-.l I~~~~~ c d 110 mv Fig. 11. Initiation and conduction of nerve excitation in the arterial preparation. Responses to three repetitive brief pulses (200,us duration, 100 V intensity and 20 ms interval) were recorded before (a and b) and after (c and d) the removal of adventitial connective tissue from a 0 5 cm region surrounding the stimulating partition. a, b and c untreated region; d treated region; a and c, at 1-5 cm from the partition; b and d at 0 2 cm from the partition. Dark zone represents the adventitial connective tissue. 2 s Control Without connective tissue Control Without endothelium _nk 20 mv Fig. 12. Effect of elimination of adventitial connective tissue (upper records) on the EJP and of removing endothelium (lower records) on the hyperpolarizing response, in the cardiac preparation. the treated region or in the untreated region, though the EJP was recordable from both regions before the treatment (n = 4) (Fig. 11). This suggests that the brief pulse stimulates nerves near the stimulating partition, which, in turn, conduct to the regions more distant from the partition. 1 s DISCUSSION The present experiments, in which the electrical responses of the smooth muscles of the coronary arteries were recorded from a preparation containing arterial and ventricular tissues, gave clear evidence that activation of cardiac muscle induces activation of the smooth muscle of the coronary artery. For the depolarizing

15 INTERACTION BETWEEN HEART AND CORONARY MUSCLE 253 response, the response recorded from the arterial preparations was strongly suggestive of an EJP and electrical stimulation applied to the isolated cardiac muscle also produced an EJP. These conclusions were obtained on the basis of the behaviour of the EJP and the hyperpolarizing response recorded intracellularly from coronary artery smooth muscles. The following possible causes of the electrical responses of the smooth muscle should be considered: (1) the potential change in the arterial smooth muscle of the cadiac preparation is due to movement artifacts; (2) the potential change in the arterial smooth muscle of the cardiac preparation is caused by extracellular potential change associated with local circuit currents induced by the action potential of ventricle muscle; (3) the potential change in the smooth muscle is evoked by a direct stimulation of the smooth muscle; (4) a brief electrical pulse directly stimulates not only cardiac muscles but also the nerves and the endothelium of the cardiac preparation. The shapes of the EJP and the hyperpolarizing response recorded from the cardiac preparation which produces strong muscle movement were similar to those recorded from the arterial preparations in which movement was small. No electrical responses were recorded from the coronary artery which was put on the ventricle as shown in Fig. 7, although the artery must be influenced by cardiac movement. These results excluded possibility (1). Possibility (2) must be considered under the conditions shown in Fig. 8. However, the influence of the electrotonic current would be greatly diminished under the standard condition of the organ bath in the present study, because the extracellular potential associated with the action potential of ventricular muscle was very small, even just at the surface of the arterial smooth muscle of the cardiac preparation; moreover, when repetitive brief stimulations with long intervals were applied to the cardiac preparations, use-dependent changes in the amplitude of the EJP and the hyperpolarizing response of the coronary artery were observed. These results excluded possibility (2). A small potential change is produced by a brief pulse when the smooth muscle of quiescent type is placed at the stimulating partition (Mekata, 1981). In the present study, the arteries of the cardiac preparations were always far from the partition, and the smooth muscles never produced an electrical response to brief pulses without an action potential in the cardiac muscle. Moreover, the time (of the order of tens of milliseconds) taken to initiate the electrical change of the smooth muscles either in the arterial or cardiac preparations is much longer than the pulse duration (of the order of tens to hundreds of microseconds). These observations excluded possibility (3) Ṗossibility (4) can be diminished by using the correct shape and arrangement of the cardiac preparation (as shown in Methods). However, a few functional nerves might remain in a cardiac preparation placed just at the stimulating plate. The following findings in the present study provide electrophysiological evidence to exclude this possibility: (i) the identical threshold for the intensity and the duration of the brief pulse for inducting an action potential in the ventricular muscle and an electrical response of the coronary smooth muscle, in all of the cardiac preparations used here; (ii) generation of the electrical responses of the smooth muscles of the cardiac preparations in an all-or-nothing manner, but in a graded manner in the

16 254 F. MEKA TA arterial preparations; (iii) single electrical response of the smooth muscles of the cardiac preparations to repetitive stimulation with short intervals, but multiple responses in the arterial preparations. There are two possibilities which may account for the transmission of the signal of the action potential of the ventricular muscle to the smooth muscle. One is that the action potential directly stimulates the arterial smooth muscle either through a chemical substance released from the ventricular muscle or by an electrotonic current induced by the action potential. The other is an indirect route where the action potential evokes a potential change through an activation of nerves or endothelium by either chemical or electrical stimulation. The possibility of a direct route perhaps can be excluded, because of the use-dependent changes of the EJP and the hyperpolarizing response that occurred, whereas direct stimulation of the smooth muscle does not show such a reduction (Mekata, 1981), and removal of the connective tissue and endothelium suppressed the EJP and the hyperpolarizing response, respectively. It is unclear whether the nerves or endothelium are activated chemically or electrically. However, there is some information for resolving this problem. Histological photographs show the existence of a 20-40,am gap between the nerves and the cardiac muscle layer in arteries having the diameters used in the present study, and perhaps nerves run more than tens of micrometres from the ventricle muscle layer. The diffusion rate of chemical substances in arterial tissues are known to be approximately ym s-' (Bevan, Bevan & Duckles, 1980). Thus, at a minimum, several hundred milliseconds are needed for the transmission of the chemical substance from the cardiac muscle to nerves. This suggests that the substance might influence the nerve or endothelium in a cumulative manner but not in the short term. The present experiments suggest that cardiac action potentials stimulate the nerves and endothelium of the artery, perhaps in an electrical manner, which in turn activates the smooth muscle. The reason why the cardiac action potential can directly stimulate the nerve and endothelial cells but not the smooth muscle of the artery may be due to differences in the values of the time constants of the membrane of these cells. The time constant of the membrane of the coronary artery was found to be comparatively large. The local circuit current induced by the cardiac action potential had the rapid deflection usually seen in electrocardiograms and suggested from Fig. 8, although the action potential itself had a prolonged duration. Therefore, the current produced by the cardiac action potential would not be effective for stimulating the smooth muscle of the coronary artery with its long membrane time constant. Although the time constants of the cell membrane of the nerve and endothelium were not measured in the present experiments, rectangular pulses of current of short duration induced EJPs and hyperpolarizing responses in the arterial preparation, suggesting that the endothelium and nerve had small time constants. In addition, small time constants have been reported for many different kinds of nerves (Tasaki, 1959). In the case of endothelial cells, although there are no reports giving the time constant of an intact cell, the membrane potential deflection of dispersed single cells associated with current application indicated a very small time constant (Busse, Fichtner, Luckhoff & Kohlhardt, 1988). Furthermore, this study found

17 INTERACTION BETWEEN HEART AiND CORONARY MUISCLE 255 anomalous rectification of the cell membrane. These electrical properties of the endothelial cell membrane suggest that electrical stimulation might readily activate this cell. Such electrical transmission of signals between cells as seen in the present experiments has been reported in studies of electrical synapses between nerves (Zachar, 1967), where a comparatively small intracellular resistance in the longitudinal direction and close contact of cells is required for successful electrical transmission. There was a wide gap between the cardiac muscle and the coronary artery in the cardiac preparation of the monkey used in the present study, and nerves run along the artery. Endothelium is further from the cardiac muscle than the nerve and smooth muscles. These factors suggest that electrical transmission of the cardiac action potential into the coronary artery may occur with difficulty. However, the results suggest that electrical transmission from the cardiac muscle to the nerve and endothelium does occur, even under such difficult conditions, which may be due to the special geometry of the coronary artery. The coronary artery is encircled with cardiac muscle, so that a large part of the concentrated local circuit currents generated by thick layers of cardiac muscle cells will flow into the coronary artery, raising the current density around the artery. The large magnitude of the local circuit current which was recorded when the solution level was low in the organ bath, and the fact that the QR amplitude in the intracoronary cardiogram was much larger than usual, support the above speculation. The tension of the artery of the cardiac preparation was not measured. Deflections of the membrane potential of smooth muscle do not always result in tension changes, as seen, for example, when a 1-15 mm-k' solution was applied (Casteels, Kitamura, Kuriyama & Suzuki, 1977). However, the tension of the smooth muscle of the monkey coronary artery has been reported to change in parallel with the membrane potential in the range more positive than about -50 mv (Mekata, 1986). Since the membrane potential measured in the present experiments was between -40 and -45 mv, the membrane potential change associated with the cardiac action potential was likely to produce a marked tension change. The author would like to thank Dr Alison Brading for critical reading of the manuscript and helpful suggestions. REFERENCES BP,NY, J.-L. & BRUNET, P. C. (1988). Electrophysiological and mechanical effects of substance P and acetvlcholine on rabbit aorta. Journal of Physiology 398, BERNE, R. Al. & LEVY, MI. N. (1981). Coronary circulation. Cardiovascular Physiology, 4th edn, pp C. V. Mosby, St Louis, MO, tjsa. BEVAN. J. A., BEVAN, R. 1). & DUCKLES, S. P. (1980). Adrenergic regulation of vascular smooth muscle. In Handbook of Physiology. section 2. The Cardiovascular System, vol. 2, Vascular Smooth.Muscle, ed. BOHR, D., SOMLYO, A. P. & SPARKS. H. VT. JR, pp American Physiological Society, XVaverly Press 13altimore. MI1). USA. BOZLER, E. (1938). Action potentials of visceral smooth muscle. American Journal of Physiology 124, BUSSE, R., FICHTNER, H.. LuC1KIIOFF. A. & KOHLHARDT, M. (1988). Hyperpolarization and increased free calcium in acetylcholine-stimulated endothelial cells. American Journal of Physiology 255. H

18 256 F. MEKATA CASTEELS, R., KITAMURA, K., KURIYAMA, H. & SUZUKI, H. (1977). Excitation-contraction coupling in the smooth muscle cells of the rabbit main pulmonary artery. Journal of Physiology 271, FEIGL, E. 0. (1983). Coronary physiology. Physiological Reviews 63, HENRY, J. P. & MEEHAN, J. P. (1971). The Circulation. Year Book Medical Publishers, Chicago, IL, USA. KEATINGE, W. R. & HARMAN, M. C. (1980). Local regulation of blood vessels by chemical agents and by intravascular pressure and flow. In Local Mechanisms Controlling Blood Vessels, pp Academic Press, New York, USA. KOMORI, K., LORENZ, R. R. & VANHOUTTE, P. M. (1988). Nitric oxide, ACh and electrical and mechanical properties of canine arterial smooth muscle. American Journal of Physiology 255, H KoMORI, K. & SUZUKI, H. (1987). Electrical responses of smooth muscle cells during cholinergic vasodilation in the rabbit saphenous artery. Circulation Research 61, MEKATA, F. (1971). Electrophysiological studies of the smooth muscle cell membrane of the rabbit common carotid artery. Journal of General Physiology 57, MEKATA, F. (1974). Current spread in the smooth muscle of the rabbit aorta. Journal of Physiology 242, MEKATA, F. (1981). Electrical current-induced contraction in the smooth muscle of the rabbit aorta. Journal of Physiology 317, MEKATA, F. (1984). Different electrical responses of outer and inner muscle of rabbit carotid artery to noradrenaline and nerves. Journal of Physiology 346, MEKATA, F. (1986). The role of hyperpolarization in the relaxation of smooth muscle of monkey coronary artery. Journal of Physiology 371, TASAKI, I. (1959). Conduction of the nerve impulse. In Handbook of Physiology, section I, Neurophysiology, vol. I, ed. FIELD, J., MAGOUN, H. W. & HALL, V. E., pp American Physiological Society, Waverly Press, Bethesda, MD, USA. ZACHAR, J. (1967). Electrophysiology of peripheral synaptic junctions. In Electrophysiological Methods in Biological Research, ed. BURES, J., PETRAN, M. & ZACHAR, J., pp Academic Press, New York.